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Langmuir 2007, 23, 694-699
Trifluoperazine Causes a Disturbance in Glycerophospholipid Monolayers Containing Phosphatidylserine (PS): Effects of pH, Acyl Unsaturation, and Proportion of PS Agnieszka Broniec,‡ Anja Underhaug Gjerde, Anne Berit Ølmheim, and Holm Holmsen* Department for Biomedicine, UniVersity of Bergen, Bergen, Norway ReceiVed June 6, 2006. In Final Form: September 28, 2006 We have studied the interaction of trifluoperazine (TFP) with monolayers of various glycerophospholipids at 37 °C. TFP (1-10 µM) had little effect on surface pressure/molecular area isotherms in monolayers (on pure water) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine but greatly increased the mean molecular area (mma) of dipalmitoylphosphatidylserine; the increment was greatest between 0 and 1 µM, and a further increase to 10 µM TFP gave only a slight increase in mma. With phosphatidylserine (PS)-containing stearoyl and varying acyls in the sn-1 and -2 positions, respectively, TFP increased the mma in a manner that depended on the number of double bonds and chain length. In mixtures of DPPC with two of these PS species the TFP-induced mma of the monolayers (on buffer, pH 7.4) increased linearly with the proportion of PS. Both PS and TFP have ionizable groups, and the TFP-induced mma increase had optima at pH 5.0 and 7.0. We conclude that the TFP-PS interaction is mainly, but not entirely, driven by electrostatic interactions between the TFP cation and PS headgroup anion, with an insertion of the phenothiazine moiety among the acyls in the monolayer that depends on the packing of the acyls.
Introduction We have previously shown that the phenothiazines chlorpromazine (CPZ) and trifluoperazine (TFP) interfere with polyphosphoinositide metabolism in stimulated platelets.1-5 Since these cells do not contain D2 receptors,6 which are assumed to be the main target for the phenothiazines,7,8 it was suggested that the phenothiazines affected polyphosphoinositide metabolism through an interference with the platelet membrane structure that changed the relative positions of the inositol lipid substrates with respect to their enzymes in the polyphosphoinositide cycle. Thus, we found that in micromolar concentrations CPZ caused large increases in the mean molecular areas of acidic but not in those of neutral glycerophospholipids in monolayers,9,10 findings that have been confirmed by others with both CPZ11-13 and TFP.12 Similar findings by us,14-17 using magic angle spinning * To whom correspondence should be addressed. E-mail: holm.holmsen@ biomed.uib.no. Phone: +47 55 58 64 44. Fax: +47 55 58 63 60. ‡ Present address: Biophysics Department, Biochemistry, Biophysics and Biotechnology Faculty, Jagiellonian University, Krakow, Poland. (1) Holmsen, H.; Daniel, J. L.; Dangelmaier, C. A.; Molish, I.; Rigmaiden, M.; Smith, J. B. Thromb. Res. 1984, 34, 419-428. (2) Holmsen, H.; Dangelmaier, C. A. Thromb. Haemostasis 1990, 64, 307311. (3) Tysnes, O.-B.; Steen, V. M.; Frølich, K. W.; Holmsen, H. FEBS Lett. 1990, 264, 33-36. (4) Frølich, K. W.; Aarbakke, G. M.; Holmsen, H. Biochem. Pharmacol. 1992, 44, 2013-2020. (5) Tharmapathy, P.; Fukami, M. H.; Holmsen, H. Biochem. Pharmacol. 2000, 60, 1267-1277. (6) Ricci, A.; Bronzetti, E.; Mannilo, F.; Mignini, F.; Morocetti, C.; Tayebati, S.; Amenta, F. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2001, 363, 376-82. (7) Seeman P. Pharmacol. ReV. 1972, 24, 583-655. (8) Dahl, S. G.; Hough, E.; Hals, P. A. Biochem. Pharmacol. 1986, 35, 12631269. (9) Agasøster, A. V.; Tungodden, L. M.; Cejka, D.; Bakstad, E.; Sydnes, L. K.; Holmsen, H. Biochem. Pharmacol. 2001, 61, 817-825. (10) Agasøster, A. V.; Holmsen, H. Biophys. Chem. 2001, 91, 37-47. (11) Jutila, A.; So¨derlund, T.; Pakkanen, A. L.; Huttunen, M.; Kinnunen, P. Chem. Phys. Lipids 2001, 112, 151-163. (12) Hidalgo, A. A.; Caetano, W.; Tabak, M.; Oliveira, O. N. Biophys. Chem. 2004, 109, 85-104. (13) Bialkowska, K.; Bobrowska-Hagerstrand, M.; Hagerstrand, H. Z. Naturforsch. 2001, 56, 826-830. (14) Nerdal, W.; Gundersen, S. A.; Thorsen, V.; Høiland, H.; Holmsen, H. Biochim. Biophys. Acta 2000, 1464, 165-75.
solid-state 13C NMR on bilayer samples with partial hydration (12 H2O molecules per phospholipid), showed that CPZ had low or no interaction in the acyl packing of liposomes made of phospholipids without a net headgroup charge and with saturated acyl chains, dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC), while it caused a large (5-15 ppm) shift to higher ppm values of ∼30% of the acyl chain carbon resonances in liposomes composed of porcine brain phosphatidylserine (PBPS) and DPPC. In general, phospholipids in bilayers with low hydration will have to share the few available water molecules among the polar groups distributed in the polar region of the bilayer. This promotes dense packing of the phospholipids and consequently lowers bilayer fluidity compared to the corresponding fluidity of a fully hydrated bilayer. Similar interactions of CPZ with glycerophospholipid bilayer vesicles have also been shown by fluorescence.18 Both TFP and CPZ are amphiphilic molecules with hydrophobic phenothiazine ring systems and positively charged (hydrophilic) tail groups, an aliphatic tail for CPZ and a piperazine group for TFP (Scheme 1). Note that three methylene groups in both phenothiazines separate the ring N and the tail N. This has proven to be important for CPZ, which interacts much more strongly with dipalmitoylphosphatidylserine (DPPS) monolayers than a CPZ analogue with two methylene groups between these two nitrogens.9 TFP has two ionizable N atoms in the piperazine ring with a pK around 8 which decreases with binding to membranes;19-23 the single tail N of CPZ has a pK of 8.624 to 9.8.19 The cmc for TFP is reported to vary from 4.2 × 10-5 (15) Gjerde, A. U.; Holmsen, H.; Nerdal, W. Biochim. Biophys. Acta 2004, 1682, 28-37. (16) Chen, S.; Gjerde, A. U.; Holmsen, H.; Nerdal, W. Biophys. Chem. 2005, 117, 101-109. (17) Song, C.; Holmsen, H.; Nerdal, W. Biophys. Chem. 2005, 120, 178-187. (18) Gjerde, A., U.; Holmsen, H. Manuscript in preparation, 2006. (19) The Merck Index, 12thth ed.; Merck & Co., Inc.: Whitehouse Station, NJ, 1996. (20) Caetano, W.; Tabak, M. J. Colloid. Interface Sci. 2000, 225, 69-81. (21) Malheiros, S. V.; dePaula, E.; Meirelles, N. C. Biochim. Biophys. Acta 1998, 1373, 332-340. (22) Malheiros, S. V.; Meirelles, N. C.; de Paula, E. Biophys. Chem. 2000, 83, 89-100.
10.1021/la061628b CCC: $37.00 © 2007 American Chemical Society Published on Web 12/01/2006
TFP Causes a Disturbance in Glycerophospholipids
Langmuir, Vol. 23, No. 2, 2007 695
Scheme 1. Structural Formulas of CPZ and TFP
Figure 1. Different phases during compression of a glycerophospholipid monolayer in the Langmuir apparatus.
10-4
to 3 × whereas that of CPZ seems to vary between 2.2 × 10-3 M20 and 1.9 × 10-2 M,26 but it is noteworthy that determination of the cmc was found to depend very much on the conditions used.21-26 A well-documented action of phenothiazines is their strong binding to and inhibition of calmodulin, and TFP is not an exception.27 TFP, also called stelazine, is classified as a conventional (“typical”) antipsychotic drug with severe side effects and has been on the market since the 1960s.28 Both CPZ and TFP are used to treat schizophrenia, but due to their many side effects (extrapyramidal effects such as parkinsonism, akathasia, and dystonia besides drowsiness, dizziness, blurred vision, dry mouth, upset stomach, and more), their use in the Western world is presently ceasing. Due to their very good therapeutic effects and low prices, CPZ and TFP are used a lot in the rest of the world (http://www.who.int/en/). As described above, TFP has many chemical properties relatively in common with CPZ and has been reported to increase the mean molecular area in monolayers of the negatively charged dipalmitoylphosphatidylglycerol but not of DPPC.12 We wanted to study the interaction of TFP with monolayers of glycerophospholipids commonly present in biological membranes and report here the effects of TFP on such monolayers where the headgroups and acyl groups have been systematically varied. We found that TFP interfered most with the ionizable PS (pK )
